Chronic exposure to tobacco smoke accounts for ~6 million deaths per year making health complications from smoking the primary cause of preventable mortality in the world. Nicotine, the addictive component of tobacco smoke, initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs), yet nicotine replacement therapy and existing smoking cessation aids that target abundant nAChR subtypes are minimally effective. Thus, a better understanding of neural circuits and neurotransmitter systems that contribute to nicotine withdrawal syndrome is needed to facilitate the development of new therapies for smoking cessation. Recent studies have identified the habenulo-interpeduncular (Hb-IPN) axis as a potentially important circuit that is hypothesized to mediate the aversive effects of high doses of nicotine. In addition, this pathway is involved in physical withdrawal symptoms in rodents. However, the role of the Hb-IPN circuit in affective withdrawal and the neurotransmitter systems underlying the manifestation of these symptoms are unknown. Our data indicate that the IPN may be a neuro-anatomical substrate for anxiety during nicotine withdrawal. In addition, preliminary data indicate that the stress peptide, corticotropin releasing factor (CRF), and its cognate receptors (CRF receptors) within the IPN play a significant role in nicotine withdrawal-induced anxiety in a mouse model of nicotine dependence. These observations provide the foundation for this application. We propose to test the hypothesis that chronic nicotine engages CRF signaling within the IPN, initiating the anxiogenic effects of nicotine withdrawal. In Aim 1 we propose to use pharmacological and viral mediated gene delivery approaches to determine if CRF receptor signaling in the Hb-IPN axis is critical for the expression of anxiety during nicotine withdrawal. In Aim 2, we will use a biophysical approach to determine how chronic nicotine and withdrawal modulates neuronal excitability within the IPN. It is anticipated that elucidating the circuitry and mechanistic detais of the anxiogenic effects of nicotine withdrawal will provide new insights into novel therapeutic strategies to facilitate smoking cessation.